Method for purifying GBS toxin/CM101

ABSTRACT

A method for purifying a polysaccharide from group B β-hemolytic Streptococcus (GBS) bacteria includes contacting a bacterial fermentation stock with a hydrophobic interaction chromatography (HIC) resin. Additional steps may include a phenol/saline extraction and an ion exchange chromatography. The method results in a product having very high purity. The product of the purification provides a composition which is highly useful in both research and therapeutic settings.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. Ser. No. 08/744,770, filed Sep.30, 1996 and issued as U.S. Pat. No. 5,811,403 on Sep. 22, 1998.

INTRODUCTION

1. Technical Field

This invention relates to improved methods of purification for apolysaccharide.

2. Background

CM101, a GBS toxin, is a pathogenic molecule isolated from group Bβ-hemolytic Streptococcus (GBS) bacteria. Newborn infants may becomeinfected with GBS, a condition known as GBS pneumonia or "early-onsetdisease," and suffer from sepsis, granulocytopenia, and respiratorydistress, i.e. pulmonary hypertension and proteinaceous pulmonary edema(Hellerqvist, C. G. et al., Studies on group B β-hemolytic streptococcusI. Isolation and partial characterization of an extra-cellular toxin.,Pediatr. Res., 15:892-898 (1981)).

Despite the harmful effects to neonates exposed to GBS, CM101 is notknown to cause toxicity in older humans. In fact, research into thistoxin has revealed a significant therapeutic application. See U.S. Pat.No. 5,010,062 and Hellerqvist, C. G. et al., Early Results of a Phase ITrial of CM101 in Cancer Patients., Proceedings of the AmericanAssociation of Cancer Research Annual Meeting (1995), wherein CM101 isutilized to inhibit vascularization of tumors. Obtaining purified CM101is critical, therefore, for both research and therapeutic purposes.

CM101 is a complex polysaccharide toxin having a molecular weight ofapproximately 300,000 Daltons and comprising N-acetyl-galactosamine,N-acetyl-glucosamine, glucose, galactose, and mannose residues.Carboxylic acid residues are also believed to be an integral part of themolecule. Repeating active epitopes most likely play an important rolein the pathophysiological response to CM101 by crosslinking receptors ontarget endothelium (Hellerqvist, C. G. et al., Early Results of a PhaseI Trial of CM101 in Cancer Patients., Proceedings of the AmericanAssociation of Cancer Research Annual Meeting (1995)).

U.S. Pat. No. 5,010,062 provides a method of purification of a GBStoxin. The method taught is labor-intensive, however, requiring numeroussteps with continual levels of loss of biological activity.

Purification of CM101 as presently known in the art provides an endmaterial which is only 40% pure as measured by chemical analyses andbiological assays. The other 60% comprises plant and yeastpolysaccharides and endogenous bacterial polysaccharides. The plant andyeast contaminants originate for the most part in the additives to thecommercial culture media used for optimal growth of the GBS bacteria.The endogenous contaminants include GBS polysaccharides including groupand type specific antigens (Paoletti, L. C. et al., Neonatal mouseprotection against infection with multiple group B streptococcal (GBS)serotypes by maternal immunization with a tetravalent GBSpolysaccharide-tetanus toxoid conjugate vaccine, Infect. Immun.62(8):3236-43 (1994); Michon, F., Multiantennary group-specificpolysaccharide of Group B Streptococcus, Biochem., 27:5341-51 (1988)).CM101 of this 40% purity level represents the current clinical grade.There is a need, therefore, for a purification method of CM101 whichresults in an end product with increased overall purity, preferably withthe removal of extraneous plant and yeast polysaccharides and GBSantigenic polysaccharides.

Additionally, the purification scheme known in the art includesenvironmentally unsound steps, such as the use of a large volume ofphenol in a phenol:water extraction. Phenol is a well-known causticmaterial.

Therefore, objects of the present invention are to provide apurification method resulting in (i) a material of high purity, (ii)using a minimal number of steps, (iii) minimizing the use of caustic ortoxic materials such as phenol, and (iv) increasing the yield ofmaterial.

SUMMARY OF THE INVENTION

The above objects have been achieved with the invention describedherein. Particularly, a purification scheme including a hydrophobicinteraction chromatography (HIC) resin for purification of CM101 fromGBS bacterial culture media results in a product of greater than 95%purity.

One aspect of this invention is a process for purifying a polysaccharidetoxin from GBS bacteria, the process including the use of an HIC resin.The present invention also includes a substantially pure polysaccharidetoxin from GBS bacteria produced by the method disclosed herein, and apharmaceutical composition comprising a substantially pure toxin and apharmaceutically acceptable carrier. The pharmaceutical composition maybe used to treat a patient having a medical condition. For example, atumor patient may be treated with the composition of the presentinvention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a CM101 purification scheme of the present invention.

FIG. 2 illustrates a known CM101 purification scheme.

FIGS. 3a-3a are quantitative hydrolysis standard curves showing the doseresponse of a PAD detector for 5 μg (FIG. 3a), 20 μg (FIG. 3b), and 50μg (FIG. 3c) of dextran (a glucose polymer) with 6-deoxy glucose as aconstant internal standard.

FIG. 4 shows the separation of standard sugar samples.

FIGS. 5a-b are elution profiles of a media concentrate on abutyl-Sepharose HIC column. FIG. 5a is measured at UV 206 absorbance.FIG. 5b is measured at UV 280 absorbance.

FIG. 6a is an HPLC profile of an HIC-purified water-eluted fractioncontaining CM101 (16 min peak) and monitored at UV 203 absorbance on aMillenium 2000 Diodo-Ray detector (Waters, Millford, Mass.).

FIG. 6b is a Diodo-Ray spectrum corresponding to FIG. 6a andillustrating minimal presence of 260 absorption (RNA and DNA) and 280absorption (tyrosine-containing protein) for the CM101 containing (16min) peak.

FIG. 7a is an elution profile monitored at 203 nm showing the purity ofthe HIC water-eluted peak of FIG. 6a further subjected to phenol/salineextraction and subsequent DEAE chromatography.

FIG. 7b is a Diodo-Ray spectrum illustrating the purity of theCM101-containing peak of FIG. 7a as evidenced by the narrow symmetricpeak and the lack of absorption at 260 nm (RNA/DNA) and 280 nm(protein).

FIG. 8 is a profile of IL-6 activity by ANA-1 Assay of fractions fromobtained from an HIC column.

FIG. 9 illustrates a sugar analysis of CM101 purified by the method ofthe present invention.

FIG. 10 is an HPLC profile of current clinical grade CM101 furthersubjected to HIC chromatography.

FIG. 11 illustrates a sugar analysis of a sample of current clinicalgrade CM101 which was further purified by HIC and HPLC.

DESCRIPTION OF SPECIFIC EMBODIMENTS

GBS toxin as used herein is defined as any fraction or componentisolated from natural or lysed GBS bacteria, or derived from mediasupernatants of lysed and/or autoclaved GBS bacteria, and which has abiological activity of evidenced by induction of respiratory distress inthe sheep assay (Hellerqvist, C. G. et al., Studies on group Bβ-hemolytic streptococcus I. Isolation and partial characterization ofan extra-cellular toxin., Pediatr. Res., 12:892-898 (1981)) oractivation of complement and binding to neovasculature as demonstratedby a peroxidase-antiperoxidase (PAP) assay of a tumor tissue specimen(Hellerqvist, C. G. et al., Anti-tumor effects of GBS toxin: apolysaccharide exotoxin from group B β-hemolytic streptococcus, J. CancRes. Clin. Oncol., 120:63-70 (1993); and Hellerqvist, C. G. et al.,Early Results of a Phase I Trial of CM101 in Cancer Patients.,Proceedings of the American Association of Cancer Research AnnualMeeting (1995)).

Substantially pure GBS toxin means a preparation in which GBS toxin isgreater than 40% pure (e.g., present in a concentration of at leastabout 40% by weight), preferably at least approximately 60% pure, morepreferably at least approximately 90% pure, and most preferably at leastapproximately 95% pure.

A source for GBS starting material for use in the method of the presentinvention may be obtained by culturing strains of Group B β-hemolyticStreptococcus bacteria that have recently infected or are capable ofinfecting newborn infants. Isolates of such strains may be obtained fromthe blood of infected infants.

High production of CM101 generally requires fermentation with thecomplex media THB which contains high molecular weight material in theform of polysaccharides and proteins for GBS optimum growth and CM101production. During the fermentation process, the bacteria produce fromthe nutrients quantities of proteins, nucleic acids, and polysaccharidesother than CM101. The estimated concentration of CM101 in thefermentation broth is less than 0.1% by weight.

The purification method of the present invention employs hydrophobicinteraction chromatography (HIC) which eliminates the bulk of theendogenous and exogenous contaminating proteins, nucleic acids, andpolysaccharides more efficiently than known methods and results in anend product which contains 10-50% pure CM101. In just one step ofcontacting the GBS starting material and the HIC resin, this representsa 100-500 fold purification from the starting material.

Use of an HIC resin for purification of a polysaccharide is surprisingand novel because HIC columns are designed for purification ofhydrophobic proteins and are not believed useful for polysaccharidesfree of proteins and lipids. Polysaccharides are generally characterizedas being hydrophilic due to their numerous hydroxyl groups. Applicationof a starting material to an HIC column under the conditions recommendedby the manufacturer and used by practitioners skilled in the art wouldtherefore be with the intention of retaining proteins and allowingpolysaccharides to pass through the column unbound.

The surprising discovery is that CM101 has hydrophobic properties thatallow use of the present purification scheme to achieve a high level ofpurity. Especially surprising is that CM101 has significantly morehydrophobic characteristics than most of the proteins andpolysaccharides present in the supernatant from which the CM101 isisolated. Greater than 98% of these protein and polysaccharidecontaminants pass through the HIC column.

Although the HIC resin is generally employed in an HIC column, this stepalternatively may be performed by contacting the resin and the startingmaterial in some other manner. For example, the GBS source and the resinmay be placed in a vessel together in a batchwise process, and thetoxin-containing portion subsequently separated from the resin as bycentrifugation.

Additional purification steps may include a phenol/saline extraction ina small volume relative to the prior methods (approximately 1000-foldreduced) and an ion exchange column. These additional purification stepscontribute to an end product with greater than 95% purity.

HIC is a method used to separate proteins, such as membrane proteins,based on their hydrophobic nature. An HIC resin is defined as a resinhaving interactive hydrophobic groups which are generally covalentlyattached to a support such that the hydrophobic groups are free tointeract with substances in contact with the resin. Examples ofhydrophobic groups include alkyl, alkoxy, and aryl groups. The preferredHIC resin to be used in accordance with the present invention has asupport with attached aliphatic groups of two or more carbons,preferably alkyl groups in the range of 2 to 12 carbons, and morepreferably normal or branched butyl groups. Phenyl groups or alkoxygroups of up to 20 carbons are also preferred interactive hydrophobicgroups. The interactive hydrophobic groups are preferably supported bySepharose (Pharmacia) or acrylamide (Toso Haas, Montgomeryville, Pa.).According to the standard procedure for use of an HIC column, thestarting material containing the protein of interest is applied to thecolumn in up to 2 M aqueous salt solution and the bound proteins arethen eluted and separated through decreases in hydrophobic interactionsby reducing the ionic strength of the developing buffer. Changes in pHand/or temperature may also be used to alter the hydrophobicinteractions.

CM101 purification from Group B Streptococcus requires obtaining abacterial culture of GBS. Bacterial innocula are incubated to late logphase in Todd Hewitt Broth (THB) modified by supplementation with 2 g/lor more of glucose and Na₂ HPO₄. As indicated in FIG. 1, the culture isthen autoclaved. CM101 is present in the supernatant of GBS fermentationcultures at a concentration of 2-15 mg/l following autoclaving. Themedia contains approximately 15 g/l of other bacterial and mediacomponents. Thus, CM101 constitutes approximately 0.01-0.1% of thecomponents in the supernatant. After autoclaving, the media is filtered.The filtrate is concentrated via a 10,000 Dalton (10k) cutoff filter.

CM101 is then purified in accordance with this invention, as shown inFIG. 1, by applying a supernatant concentrate or reconstituted alcoholprecipitate thereof made 2 M in potassium phosphate or another salt,such as sodium phosphate, sodium or potassium sulphate, chloride, oracetate, to an HIC column preferably equilibrated in the same salt ofthe same molarity. The method wherein the media concentrate, or 10k,starting material is employed without alcohol precipitation andreconstitution is preferred because the media concentrate startingmaterial provides higher yield of CM101 than does the reconstitutedalcohol precipitate.

The CM101-containing starting material is applied to the HIC column andwashed with aqueous 2 M phosphate. Following a 2 M wash, the column isfurther developed with 1 M and 0.25 M salt, preferably phosphate. In thepreferred embodiment, the CM101 is eluted from the column with water asa single peak containing 10-50% CM101. Alternatively, water is replacedfor CM101 elution from the HIC column with 10 mM phosphate, pH 6.8 in10% ethanol in water (Buffer A), followed by 20% ethanol in water. CM101activity is recovered in both the Buffer A and 20% ethanol fractions.Use of Buffer A is generally not sufficient to remove all the CM101 fromthe HIC column, so the Buffer A wash is followed by an additional 20%ethanol wash. However, in scale-up, the ethanol constitutes anenvironmental hazard and the subsequent phenol/saline extraction of thewater peak or the Buffer A and 20% ethanol peak fractions yields CM101of approximately equal purity. The HIC procedure removes better than 98%of both the proteins and media polysaccharides remaining in the 10kconcentrate or the reconstituted alcohol precipitate.

The enriched CM101 from the HIC column may be further purified by anextraction in phenol and an aqueous salt solution, preferably 0.05 Msaline. This additional step provides a CM101 fraction of approximately95% purity.

The water or the combined Buffer A and 20% ethanol fractions eluted fromthe HIC column are either dialyzed against water and lyophilized andreconstituted in 0.05M saline or dialyzed against saline afterconcentration. Typically, phenol is added to the material and thesolution is rapidly heated to 70-80° C. When a single phase forms, thesolution is chilled to 4° C. The resulting saline phase of thephenol/saline extraction contains CM101 and may then be applied to acation exchange column, such as DEAE.

For the DEAE column procedure, the DEAE column is equilibrated in waterand then washed with 0.1 M saline, 0.05 M NaOAc, pH 7.4 and developedwith a step gradient to 0.34 M NaCl. Elution of CM101 is monitored andquantitated with an ANA-1 assay. As with the HIC resin step, an ionexchange resin may be contacted with the toxin-containing materialthrough use of equipment other than a column. The CM101-containingfraction is then dialyzed against water and lyophilized. After thephenol/saline extraction and ion exchange steps, CM101 is greater than95% pure.

The column eluates, or material resulting from the resin contact steps,are assayed for biologic activity with the ANA-1 assay. The biologicalactivity is then confirmed with a sheep assay. Table 1 depicts severalseparations of AP and 10k material obtained from different batches ofstarting materials and applied to the HIC column. Removal of denaturedprotein and media polysaccharides and other material is similar.

Although the preferred order of purification is to perform the HIC step,followed by the phenol/saline extraction, and then the ion exchangestep, purification may also be performed in another order.

FIG. 2 presents an example of a known method of CM101 purification.Notably, a 70% ethanol precipitate step is used, followed soonthereafter with a phenol/water extraction. The large volumes of ethanoland phenol required at these early stages of the known purificationmethod represent environmentally unsound practices. The methodrepresented in FIG. 2 also requires an ion exchange column, a gelfiltration column, and a lentil lectin column.

The prior method contains numerous steps, including environmentallyhazardous ones. On the other hand, the method of the present inventionis effective, gives higher purity and 2 to 25 times the yield, andminimizes use of environmentally unsound materials.

The environmentally hazardous phenol-water extraction step is reduced1000-fold as compared to the previously used procedures. Furthermore,additional purification as by a gel filtration procedure is eliminated.The lentil lectin chromatography step of the prior method is alsodeleted. The end product of the HIC column, phenol/saline extraction,and ion exchange column steps has approximately 95% purity, so othertreatments are unnecessary.

The CM101 purified by the method of the present invention may be usedfor research or therapeutic purposes. The CM101 is particularly usefulwhen combined with a pharmaceutically acceptable carrier, e.g.,reconstituted in saline and administered to a patient intravenously.Other dosage forms to administer purified CM101 may also be used. Thepharmaceutical composition of this invention comprises the substantiallypure GBS toxin of this invention in combination with a pharmaceuticallyacceptable carrier. In general, the carrier will be one that is readilymixed with the toxin to form a composition that is administrable byintravenous (IV) means. Thus, the carrier is preferably water, which mayhave other pharmaceutically acceptable excipients included to ensure itssuitability for intravenous administration. The resulting compositionwill be sterile and will have acceptable osmostic properties. Ingeneral, a suitable IV formulation is prepared in accordance withstandard techniques known to one of skill in the art. For example,Chapter 85 entitled "Intravenous Admixtures" by Salvatore J. Turco inthe Eighteenth Edition of Remington's Pharmaceutical Sciences, MachPublishing Co. (1990), incorporated herein by reference, providesstandard techniques for preparing a pharmaceutically acceptable IVcomposition useful in accordance with this invention.

Additionally, a patient having a medical condition which is found torespond advantageously to CM101 may be treated with a pharmaceuticalcomposition of the present invention. For example, a patient having atumor may be advantageously treated by intravenously administering thepharmaceutical composition taught herein. U.S. Pat. No. 5,010,062discusses the treatment of certain tumors in humans and is incorporatedherein by reference.

Quantitative and Qualitative Analysis

HPLC Analysis

The purity and amount of CM101 obtained from a sample after HICchromatography is established by high pressure liquid chromatograph(HPLC) gel filtration analysis. The gel filtration column is typicallyequilibrated with an ammonium acetate (NH₄ OAc) buffer, pH 8.4, and thebiologically active CM101 is eluted as an included homogeneous narrowpeak.

A typical detector response (UV 203 absorption) using 30, 50, and 100 μgpure CM101 standards injected in 100 μl of developing buffer on aHydragel 1000 column (Waters, Millford, Mass.) is 26×10⁶, 48×10⁶, and97×10⁶ area units, respectively which yields a dose response curve forquantitation of unknown samples.

Amino Acid Analysis

Quantitative and qualitative automated amino acid analysis may beperformed with standard commercially available equipment, e.g., PicoTag,available from Waters, Millford, Mass.

ANA-1 Assay

To monitor the biological activity of the different fermentation andpurification steps, an in vitro assay employing a transformed mousemacrophage cell line may be used. The assay measures IL-6 production ofthe mouse macrophage ANA-1 in response to CM101 exposure.

Particularly, CM101 induces raf/myc transformed murine bone marrowmacrophage cell line ANA-1 to respond in vitro by IL-6 production. Othermacrophage-like cell lines and fresh peripheral blood leukocytes canalso be used.

To perform the ANA-1 assay, samples are first diluted to the appropriaterange (depending on the expected level of CM101 activity) and four toeight concentrations are tested at 1:4 dilutions. A CM101 standard curveusing clinical grade CM101 reconstituted in PBS is generated. A 4000ng/ml solution, which gave a 2000 ng/ml final concentration after thecells were added, was made in PBS, along with six serial 1:2 dilutions.Cells at a concentration of 2×10⁶ /ml may be used, for example.Sensitivity of the assay was increased by adding 200 U/ml murine IFN-γto the ANA-1 cells. Final cultures were 100 U/ml IFN-γ.

The microtiter plate with cultures should be placed in a 37°, 5% CO₂-in-air, humidified incubator overnight (16-18 hours), and then befollowed by an ELISA IL-6 Assay (R. D. Systems, Minneapolis, Minn.).Specifically, culture supernatants are transferred to the IL-6 assayplate and the plate is held at 4° C. until the IL-6 assay is complete.

Sheep Pulmonary Arterial Pressure Assay

The toxin affects sheep lungs by increasing pulmonary hypertension,manifested by increased pulmonary arterial pressure and by increasedlung vascular permeability.

CM101 samples in phosphate buffered saline (PBS) may be administered tolambs by infusion and changes in pulmonary arterial pressure recorded at15 minute intervals. These changes in pressure are correlated to CM101activity. (Hellerqvist, C. G. et al., Studies on group B β-hemolyticstreptococcus I. Isolation and partial characterization of anextra-cellular toxin., Pediatr. Res., 15:892-898 (1981)).

Sugar Analysis

A 100 μg quantity of a sample is hydrolyzed for two hours at 100° C. ina mixture of trifluoroacetic acid (TFA), acetic acid (HOAc) and water ina ratio of 5:70:25. The solution is evaporated and the sample is furtherhydrolyzed for two hours at 100° C. in a mixture of TFA and water in aratio of 2:8. This process completely hydrolyzes all glycosidic linkagesin the sample. The N-acetyl groups originally present on the aminosugars are also removed.

The samples are then analyzed on the Dionex sugar analysis system usinga PAD (Pulsed Amperometric Detection) detector. The resolution isillustrated in FIG. 4.

The purity of the sample is established by quantitative and qualitativesugar analysis. The principle is illustrated in FIGS. 3a-3c and FIG. 4.A sample of polysaccharide quantitated by HPLC is supplemented with aninternal standard 6-deoxy-D-glucose hydrolyzed and analyzed. The methoddescribed in this section gives a linear dose response in the rangetested and qualitative analysis is accomplished by comparing retentiontimes of unknowns with the standards.

EXAMPLES Example 1

A scaled-up purification scheme for CM101

A Group B Streptococcus isolate Type III working stock was used inconjunction with a 3,000 gallon fermentor. A 25 ml seed of the bacterialculture is used for an 80 liter vessel with a 65 liter working volume(lwv) which is then used to inoculate a 750 lwv vessel, and which, inturn, goes into the final 7500 lwv (3,000 gallon) fermentor.Alternatively, the 65 lwv may be used to innoculate the 7500 lwvfermentor directly.

The cultures are terminated at late log phase by autoclaving. Thebacteria are then removed by continuous centrifugation at 10,000×g,followed by 0.45 micron cassette filtration (Millipore Corporation,Bedford, Mass.).

The resulting culture supernatant is then concentrated 15-fold throughcassette filtration using 10 kD cut off cassettes (Millipore) to 500liters. The concentrated material is then made 2 M in salt, preferablysodium phosphate, pH 7.4 (loading buffer) by dialysis.

The concentrated supernatant is then subjected to hydrophobicinteraction chromatography, through the use of a 60 liter n-butylSepharose column (Pharmacia, Uppsala, Sweden) using a BioPilot system(Pharmacia). The capacity of the n-butyl Sepharose resin for thebiologic CM101 activity in the media concentrate with no flow-through ofactivity is approximately 80 liter of media to one liter of resin. Afterthe concentrated supernatant is loaded onto the column, the column iswashed with the loading buffer followed by 1 M and 0.25 M phosphatebuffer, pH 7.4. The CM101-containing fraction is eluted with water inapproximately 120 liters or two column volumes and concentrated to 2liters in a 10 kD cut-off cassette. The column elution is controlled bya preestablished program in the BioPilot and the eluate is monitored byUV absorption at 206 and 280 nm, conductivity, and pH.

The CM101-containing 2 liter fraction is dialyzed against 0.05 M saline,pH 7.0 and then heated to the range of 75-80° C. and 0.2-2 liters ofphenol are added. The mixture is then heated to 80° C. and maintained atthat temperature for 5 minutes. Following this, the mixture is chilledto 4° C. The water phase resulting from this step is applied to a DEAESephacel FF column (Pharmacia, Uppsala, Sweden) equilibrated in water.The column is washed with 100 mM saline, 0.05 M NaOAc, pH 7.4, and thebiologically active material, CM101, is then eluted from the DEAE columnwith a NaCl gradient. The biological activity is detected by Il-6 assayand HPLC analysis. The quality of the CM101 purified through thisprocedure is established by HPLC and sugar analysis as well asbiological activity assays by Il-6 and sheep tests.

This scaled up purification scheme provides the advantage of avoidingthe large volume, early phenol-water extraction procedure of the alcoholprecipitate used in the previous procedure.

Results

FIGS. 5a-b show elution profiles of a media concentrate on abutyl-Sepharose HIC column in 2 M K₂ HPO₄, pH 7.2. The various peaks arethe results of timed step-wise changes in the elution gradient. FIG. 5arepresents the profile measured at UV 206 absorbance, which quantitatesthe peak fractions for total organic material, and shows the CM101 inthe last narrow peak (approximately 383 minutes). FIG. 5b represents theprofile measured at UV 280 absorbance, which quantitates the amount ofprotein in the different fractions.

By performing the HIC column step, CM101 is caused to bind to the columnwhereas up to 99.7% of the protein and up to 98.5% of neutral andcharged polysaccharides pass through the column, as indicated in Table1.

                  TABLE 1    ______________________________________    Purification of CM101 Activity by HIC Chromatography    Quantitation by Integration of UV 280 and 206 Profiles    Final Elution Possible Protein UV280                          Total Organic UV206                 Recovered %                          Recovered %    ______________________________________    AP 6P6   Water     0.85       2.67    AP 2P9   Water     1.08       0.19    10K5P6   Water     0.82       1.05    10K5P6   Water     0.46       2.43    AP 1 P9  Buffer A  0.39       1.90    10K5P6   Buffer A  0.50       1.51    AP 6P6   Buffer A  0.19       1.35    ______________________________________

In Table 1, different fermentation lots as alcohol precipitates (AP),AP1, AP2, and AP6, and 10k concentrates were subjected to HICchromatography and eluted with either water or Buffer A. Both processesyield approximately the same efficacious removal of exogenous andendogenous protein (UV 280) and polysaccharides and general organics (UV206).

FIGS. 6a-b present an HPLC profile, and a Diodo-Ray spectrum, of anHIC-purified water-eluted fraction containing CM101 and monitored at UV203 absorbance. These figures illustrate the minimal presence of 260absorption (RNA and DNA) and 280 absorption (protein) for the CM101containing peak.

After the HIC fraction is further subjected to the phenol/salineextraction and ion exchange steps, the purity of the HIC water-elutedpeak is further improved, as seen in FIGS. 7a-b. Note the narrowsymmetric peak and the lack of absorption at 260 (RNA/DNA) and 280(protein).

These elution profiles as well as the biological activity are similar tothose obtained when the alcohol precipitate is used as the startingmaterial for the HIC column.

The ability of the HIC fractions from the 10k starting material toinduce IL-6 synthesis in ANA-1 cells is illustrated in FIG. 8. HICchromatography yielded an approximate recovery of 50% of the totalbiologic activity in the media supernatant as measured by an ANA-1Assay.

The different fractions obtained from the 10k concentrate after HICchromatography were also tested in the sheep model for biologicactivity. The amount of CM101 activity is determined based on a doseresponse curve using current clinical CM101 (1 Unit of activitycorresponds to 7.5 μg/kg). The results are shown in Table 2 wherein HICfractionations of alcohol precipitate (AP) and media concentrate (10k)are compared.

                  TABLE 2    ______________________________________    Biologic Activity in Sheep of Fractions Obtained from HIC    Chromatography of AP and 10K Material             Alcohol Precipitate (AP)                             Media Concentrate (10k)    Fraction CM 101 Activity μg/l                             CM 101 Activity μg/l    ______________________________________    Pre-Load 466             Not Available    1M Phosphate             118              209    0.25 Phosphate              28             2970    Water    225             7520    ______________________________________

The product yield of the method of the present invention is alsoevidenced above, as the known methods provide about 300 μg of CM101 perliter of fermentation volume, as compared with the 7520 μg/l value shownabove.

The purified CM101 illustrated in FIGS. 7a-b obtained by the process ofthe present invention was also subjected to sugar analysis. The sugaryields are shown in FIG. 9.

Quantitatively, the CM101 obtained by the method of the presentinvention is greater than 95% pure carbohydrate and contains less than5% of protein established by quantitative and qualitative as presentedabove and by automated amino acid analysis (PicoTag, Waters, Millford,Mass.).

Example 2

Comparison of Current Clinical Grade and New Composition

The CM101 obtained by the method of the present invention is improvedover the current clinical grade CM101. Particularly, the HPLC elutionprofile of FIG. 10 as compared with FIG. 7a illustrates higher purity inthe sample produced according to the present invention. FIG. 7a showsone narrow and symmetric main peak, instead of several peaks.

To further demonstrate the advantageous use of the HIC column and toprovide further evidence of purification of the toxin known as CM101,current clinical grade CM101 was subjected to an HIC column and HPLCpurification and a sugar analysis was performed. The results, in FIG.11, may be compared to FIG. 9. The sugar analysis shows quantitativelyand qualitatively similar end products. This result is borne out inTable 3, as well.

                  TABLE 3    ______________________________________    Carbohydrate Composition of CM101 (Presented as Integral Carbohydrate    Ratios)            CM101                Current Clinical            Purified With                     Current Clinical                                 Grade CM101            Present Method                     Grade CM101 Further Purified    ______________________________________    Sugar    Rhamnose  0          3           0    Mannose   1          3           1    Galactose 3          24          3    Glucose   1          7           1    Glucosamine*              1          13          1    Galactosamine*              1          5           1    ______________________________________     *Present as "Nacetyl-glucosamine and Nacetyl-galactosamine", respectively     in the native polysaccharide.

Thus, the method of the present invention provides an improved method ofpurification which minimizes the difficulty of hazardous steps andprovides excellent purity. Additionally, the product produced by themethod taught herein is improved over the currently available CM101.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

What is claimed is:
 1. A method of purifying a polysaccharide toxin fromgroup B β-hemolytic Streptococcus (GBS) bacteria, which methodcomprises:(a) contacting an aqueous mixture containing the toxin with ahydrophobic interaction chromatography (HIC) resin; and (b) separatingthe toxin from the HIC resin.
 2. The method of claim 1, wherein thepurity of the toxin resulting from the HIC resin contacting step isincreased approximately 100-500 fold relative to the purity of the toxinin the aqueous mixture.
 3. The method of claim 1, wherein the toxinresulting from the step of separating the toxin from the HIC resin isgreater than 40% pure.
 4. The method of claim 1, further comprisingextracting the toxin of step (b) with an aqueous phenol mixture to forman aqueous phase including the toxin.
 5. The method of claim 4, whereinthe toxin resulting from the step of extracting the toxin with anaqueous phenol mixture is at least approximately 60% pure.
 6. The methodof claim 5, wherein the toxin resulting from the step of extracting thetoxin with an aqueous phenol mixture is at least approximately 90% pure.7. The method of claim 4, wherein the toxin resulting from the step ofextracting the toxin with an aqueous phenol mixture is at leastapproximately 60% pure.
 8. The method of claim 7, wherein the toxinresulting from the step of extracting the toxin with an aqueous phenolmixture is at least approximately 90% pure.
 9. The method of claim 1,wherein the HIC resin further comprises a resin having hydrophobicgroups selected from the group consisting of alkyl, alkoxy, and arylgroups.
 10. The method of claim 9, wherein the hydrophobic groups arealkyl groups having in the range of 2 to 12 carbons.
 11. The method ofclaim 10, wherein the hydrophobic groups are butyl groups.
 12. Themethod of claim 11, wherein the hydrophobic groups are normal butylgroups.
 13. The method of claim 9, wherein the hydrophobic groups arephenyl groups.
 14. The method of claim 1, further comprising:(c)contacting the toxin from step (b) with an ion-exchange resin; and (d)separating the toxin from the ion-exchange resin.
 15. The method ofclaim 14, wherein the toxin resulting from the step of separating thetoxin from the ion-exchange resin is at least approximately 60% pure.16. The method of claim 15, wherein the toxin resulting from the step ofseparating the toxin from the ion-exchange resin is at leastapproximately 90% pure.
 17. The method of claim 1, further comprisingafter the step of separating the toxin from the HIC resin:(c) extractingthe toxin with an aqueous phenol mixture to form an aqueous phaseincluding the toxin, (d) contacting the toxin of step (c) with anion-exchange resin; and (e) separating the toxin from the ion-exchangeresin.
 18. A method of purifying a polysaccharide toxin from group Bβ-hemolytic Streptococcus (GBS) bacteria comprising:(1) applying asource of GBS bacterial toxin to a hydrophic interaction chromatography(HIC) column, (2) eluting the HIC column to obtain an HIC eluateincluding the toxin, (3) extracting the HIC eluate with a combination ofphenol and an aqueous solution to form an aqueous phase including thetoxin, (4) applying the aqueous phase to an ion exchange column, and (5)eluting the ion exchange column to obtain an ion exchange eluate,wherein the ion exchange eluate comprises a substantially pure toxin.19. The method of claim 18 wherein the substantially pure toxin isCM101.
 20. The method of claim 18 wherein the substantially pure toxinis at least approximately 60% pure.
 21. The method of claim 20 whereinthe substantially pure toxin is at least approximately 95% pure.